In vivo NMR as a tool for probing molecular structure and dynamics in intact Chlamydomonas reinhardtii cells

Solid state NMR/Biophysical Organic Chemistr

Conformational Dynamics of Light-Harvesting Complex II in a Native Membrane Environment

Photosynthetic light-harvesting complexes (LHCs) of higher plants, moss, and green algae can undergo dynamic conformational transitions, which have been correlated to their ability to adapt to fluctuations in the light environment. Herein, we demonstrate the application of solid-state NMR spectroscopy on native, heterogeneous thylakoid membranes of Chlamydomonas reinhardtii (Cr) and on Cr light-harvesting complex II (LHCII) in thylakoid lipid bilayers to detect LHCII conformational dynamics in its native membrane environment. We show that membrane-reconstituted LHCII contains selective sites that undergo fast, large-amplitude motions, including the phytol tails of two chlorophylls. Protein plasticity is also observed in the N-terminal stromal loop and in protein fragments facing the lumen, involving sites that stabilize the xanthophyll-cycle carotenoid violaxanthin and the two luteins. The results report on the intrinsic flexibility of LHCII pigment-protein complexes in a membrane environment, revealing putative sites for conformational switching. In thylakoid membranes, fast dynamics of protein and pigment sites is significantly reduced, which suggests that in their native organelle membranes, LHCII complexes are locked in specific conformational states

Protein and lipid dynamics in photosynthetic thylakoid membranes investigated by in-situ solid-state NMR

Solid state NMR/Biophysical Organic Chemistr

Seroepidemiologic study of hepatitis B in Kouhdasht and Sepid dasht districts in 2012

Background: Hepatitis is a disease causing inflammation of the liver and impairment of its function .It is often transmitted through contaminated blood and blood products and may become chronic presenting with flu like symptoms .Also fatigue , loss of appetite , myalgia and arthralgia , nausea and vomiting are its remarkable symptoms. The aim of this research was to study the prevalence rate of hepatitis B in rural papulation of Garab, Padervand, Chaghabal and Tang-e Panj in terms of age, sex, job… and its comparison with prevalence rate in similar regions of our province and country. Materials and Methods: Aimed to determine the seroprevalence of HbsAg infection in areas of Garab, Padervand , Chaghabal and Tang-e Panj and relationship between defined epidemiological factors and the infection in the population under study, after providing a questionnaire containing the symptoms of the disease, the data were collected and blood specimens were obtained. Then the specimens were tested by ELISA . Results : Out of a total of 454 cases 227 were male and 227 were female ( identical sex ratio ) of them 27 cases (6.2 % ) were HBsAg positive . Out of infected persons 14 were male(51.9%) and 13 were female(48.1%) . Most infected ones were 20 to 40 years old and housewives , truck drivers and students were amongst the most infected cases . Conclusion : Based on the findings of the study heptitis B infection in these areas is far more frequent than the mean national rates amounting to 2 to 3 percent that is in accordance with surveillance system data in the mentioned areas . Although the findings does not define the main transmission pattern of the disease , when age and gender distribution of infected cases is considered ( almost equal sex ratio and preponderance of married ones ) we can state that this pattern is largely directed to sexual transmission .Although all age and sex groups are at risk , men and those at the age of 20 to 40 are more prone to infection . Occupation, blood transfusion , household dimension , vaccination history were significantly related to HbsAg infection

Protein dynamics and lipid affinity of monomeric, zeaxanthin-binding LHCII in thylakoid membranes

The xanthophyll cycle in the antenna of photosynthetic organisms under light stress is one of the most well-known processes in photosynthesis, but its role is not well understood. In the xanthophyll cycle, violaxanthin (Vio) is reversibly transformed to zeaxanthin (Zea) that occupies Vio binding sites of light-harvesting antenna proteins. Higher monomer/trimer ratios of the most abundant light-harvesting protein, the light-harvesting complex II (LHCII), usually occur in Zea accumulating membranes and have been observed in plants after prolonged illumination and during high-light acclimation. We present a combined NMR and coarse-grained simulation study on monomeric LHCII from the npq2 mutant that constitutively binds Zea in the Vio binding pocket. LHCII was isolated from 13C-enriched npq2 Chlamydomonas reinhardtii (Cr) cells and reconstituted in thylakoid lipid membranes. NMR results reveal selective changes in the fold and dynamics of npq2 LHCII compared with the trimeric, wild-type and show that npq2 LHCII contains multiple mono- or digalactosyl diacylglycerol lipids (MGDG and DGDG) that are strongly protein bound. Coarse-grained simulations on npq2 LHCII embedded in a thylakoid lipid membrane agree with these observations. The simulations show that LHCII monomers have more extensive lipid contacts than LHCII trimers and that protein-lipid contacts are influenced by Zea. We propose that both monomerization and Zea binding could have a functional role in modulating membrane fluidity and influence the aggregation and conformational dynamics of LHCII with a likely impact on photoprotection ability.NWO723.012.103Solid state NMR/Biophysical Organic Chemistr

Conformational Dynamics of Light-Harvesting Complex II in a Native Membrane Environment

Photosynthetic light-harvesting complexes (LHCs) of higher plants, moss, and green algae can undergo dynamic conformational transitions, which have been correlated to their ability to adapt to fluctuations in the light environment. Herein, we demonstrate the application of solid-state NMR spectroscopy on native, heterogeneous thylakoid membranes of Chlamydomonas reinhardtii (Cr) and on Cr light-harvesting complex II (LHCII) in thylakoid lipid bilayers to detect LHCII conformational dynamics in its native membrane environment. We show that membrane-reconstituted LHCII contains selective sites that undergo fast, large-amplitude motions, including the phytol tails of two chlorophylls. Protein plasticity is also observed in the N-terminal stromal loop and in protein fragments facing the lumen, involving sites that stabilize the xanthophyll-cycle carotenoid violaxanthin and the two luteins. The results report on the intrinsic flexibility of LHCII pigment-protein complexes in a membrane environment, revealing putative sites for conformational switching. In thylakoid membranes, fast dynamics of protein and pigment sites is significantly reduced, which suggests that in their native organelle membranes, LHCII complexes are locked in specific conformational states

Fractionation platform for target identification using off-line directed two-dimensional chromatography, mass spectrometry and nuclear magnetic resonance

The unambiguous identification of unknown compounds is of utmost importance in the field of metabolomics. However, current identification workflows often suffer from error-sensitive methodologies, which may lead to incorrect structure annotations of small molecules. Therefore, we have developed a comprehensive identification workflow including two highly complementary techniques, i.e. liquid chromatography (LC) combined with mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (NMR), and used it to identify five taste-related retention time and m/z features in soy sauce. An off-line directed two-dimensional separation was performed in order to purify the features prior to the identification. Fractions collected during the first dimension separation (reversed phase low pH) were evaluated for the presence of remaining impurities next to the features of interest. Based on the separation between the feature and impurities, the most orthogonal second dimension chromatography (hydrophilic interaction chromatography or reversed phase high pH) was selected for further purification. Unknown compounds down to tens of micromolar concentrations were tentatively annotated by MS and structurally confirmed by MS and NMR. The mass (0.4–4.2 μg) and purity of the isolated compounds were sufficient for the acquisition of one and two-dimensional NMR spectra. The use of a directed two-dimensional chromatography allowed for a fractionation that was tailored to each feature and remaining impurities. This makes the fractionation more widely applicable to different sample matrices than one-dimensional or fixed two-dimensional chromatography. Five proline-based 2,5-diketopiperazines were successfully identified in soy sauce. These cyclic dipeptides might contribute to taste by giving a bitter flavour or indirectly enhancing umami flavour.</p